Weirdest eye of water flea

Starshade · Post by **Starshade** » Sun Apr 01, 2012 11:23 pm

Found that among my old photos. My guess is Simocephalus vetulus. Not sure about the green tint and this bizarre eye.

Visit http://notes-from-dreamworlds.blogspot. ... asion.html for larger version of the image (the second one).

Clado · Post by **Clado** » Mon Apr 02, 2012 5:13 am

Hello Starshade,

This is a very nice picture of an ephippial female Water Flea.
The genus is not Simocephalus but Moina. I am not sure about the species. If the ephippium is reticulated entirely it might be Moina micrura.

Regards

Clado

Starshade · Post by **Starshade** » Mon Apr 02, 2012 6:02 am

Thank you Clado,

I can't tell more about the structure of the ephippium, aside from what I see on the pictures. But I was looking at pics in the internet and my flea looked exactly like Moina micrura, though I understand that Moina species might be having small features that I simply did not point attention on.

Among the small number of cladocera species that I've seen, they have the most interesting appearance, but they are very hard to photograph. When they stop moving, they tend to put their antennae under the shell, or just take another "uncomfortable" and unnatural position.

dariuskersulis · Post by **dariuskersulis** » Mon Apr 02, 2012 7:55 am

Did you use incident light here at all or all transmitted? I'm very much a newbie and trying to understand how you do it. Love this one.

Starshade · Post by **Starshade** » Mon Apr 02, 2012 9:47 am

Just transmitted light, no flash, no special tricks... simply darkfiled with crossed polarizers and careful alignment.

dariuskersulis · Post by **dariuskersulis** » Mon Apr 02, 2012 10:23 am

Starshade wrote:Just transmitted light, no flash, no special tricks... simply darkfiled with crossed polarizers and careful alignment.

How do you get polarized light though? Wondering if I could use my DIC setup to get polarized light effect.

Below the condenser annulus wheel I have a fixed prism:

And then the nosepiece prism:

Which one I should use to get the effect? Also any tips on aligning it?

And my darkfield is anything but dark, but what mostly bothers me is that with 10x objective I get a lit circle of about 1/2 diameter of the FOV and then the light sharply drops off. I've tried opening the diaphragm in the microscope base to the maximum but the illuminated circle is still way smaller than the FOV. Do I need different condenser or just the darkfield annulus? Even the circle that is lit is uneven, if I remember correctly the light drops off in some weird angle.

I think I'll use darkfield mostly with 10x and 4x objectives.

Please advise.

Starshade · Post by **Starshade** » Mon Apr 02, 2012 11:29 am

To be honest I am not familiar with your microscope and from pictures alone I can't figure all sorts of details to answer your question. I was kind of "addicted" to Zeiss gear... mostly because I didn't have access to other scopes in Germany and Russia.

Before I try to give an advice, do you have a setup like this one: http://www.microscopyu.com/articles/dic ... onfig.html? Most importantly, do you have a separate analyzer that is independent from the DIC objective prism? Also, can you somehow remove that condenser lens or replace it with an alternative one? I think you small field of view at 10x is caused by it. Can you rotate the polarizer to change the angle of polarization? Can you move the condenser up and down? Did you perform the koehler illumination steps? Without knowing such things I would not be able to give any advice, but I'm also hoping there's someone in this forum who is familiar with your kind of scopes.

By the way, you don't need dark field annulus. As long as you can reproduce the plain polarization technique, you should be able to select the right exposure time to make the dimmer background absolutely black. At least it works for me, sometimes I even forget with which settings I take pictures - that's how similar the effects can be if you know what you are doing.

dariuskersulis · Post by **dariuskersulis** » Mon Apr 02, 2012 12:03 pm

1. I think that's exact setup I have... Light > Linear polarizer > Condenser Nomarski prism > Objective > Analyzer. Except I believe my condenser diaphragm is below the Nomarski prisms and not above like in the image. But then if using darkfield Nomarski prisms don't apply.

2. When you say "separate analyzer that is independent from the DIC objective prism" do you refer to the nosepiece analyzer seen in 2nd picture. It is kind of independent, you can move the analyzer prism sideways by rotating the right knob seen on the slider. When you rotate the light changes to different colors. But I believe effect is only achieved if using Nomarski prisms in the condenser. I don't believe I noticed any difference using it in the darkfield.

3. There are screws on the condenser and I'm pretty sure I should be able to replace the darkfield stop/annulus. So should I go with smaller or larger obstruction of the dakfield stop to increase the FOV?

4. The linear polarizer (seen in 1st photo) in the condenser is unfortunately fixed and can't be rotated.

5. Yes I can move the condenser up/down and laterally.

6. Yes I went through koehler illumination steps. Focus on the subject > Close the diaphragm > Focus condenser > open the diaphragm so it just touches the FOV of 10x objective.

Starshade · Post by **Starshade** » Mon Apr 02, 2012 1:22 pm

Ok, some parts are clear, still I am missing an important element - the Nomarski prism that has to be after the objective but before the analyzer, at least that's what Zeiss gear has.
From what you are describing it appears that the DIC prism is combined with analyzer, which leaves me confused about how to use it as analyzer alone. Do I get it right? But even though I am missing this bit, as long as you have 2 polarizing elements, you can use the technique even if it will involve some duct tape fixing and buying separate filters.
That was regarding 1 and 2.

3. Again, your are not speaking Zeiss=) Apparently Nikon has the same design, so: http://www.microscopyu.com/articles/pha ... igure1.jpg Does your condenser anyhow resemble that? Does the element that is called "condenser top lens" move? Can you put it aside, unscrew, or replace? I might be wrong here, but I think it is that element that constricts the filed of view in your particular case, not an annulus which is supposed to be under the top lens. At least in my scopes I can't constrict the FOV with an annulus.

4. that sucks, but I believe you can make up for it with a rotatable analyzer, if you install one instead of the nosepiece prism that you have. Again, that's not difficult, nor expensive if I am not missing something in your scope.

5. Good, it's not a microscope for aliens at least=) You possible will need to play around with it when adjusting illumination for particular lens and particular object. By the way, if you move the condenser down, does the FOV increase?

6. So, I am almost sure you did it right, but to be 100% percent that I am getting what you saying correct: when you shut the diaphragm, is the small hole in the middle situated exactly in the middle of the FOV?

Charles Krebs · Post by **Charles Krebs** » Mon Apr 02, 2012 2:17 pm

Daniel,

A very "alien" looking creature indeed! Nicely done!
Enjoyed the video on your site very much as well.

Darius,
To get simple cross-polarized light you will need to remove the upper DIC "slider" and use a plain linear polarizer in its place. For simple polarization effects you need one polarizer somewhere beneath the subject, and a second (the "analyzer") somewhere above it. It is essential to be able to alter the rotational angles angle between these polarizers. Since the polarizer in your condenser is in a specific fixed position (the position required for the type of DIC used in your scope) you may find it easier to place a (linear) polarizing filter over the light port on the microscope base. If you pull the upper DIC slider out, you could make up a "dummy" insert that also had a linear polarizer in/on it. (Such specific pieces were undoubtedly available for your microscope but finding them may be very difficult and/or expensive). In addition to regular camera (linear not the more common "circular") polarizers there is also polarizing material available from optical supply companies such as Edmund Optical. http://www.edmundoptics.com/products/di ... uctid=1912

Many "camera" polarizers do not provide as good a cross-polarized effect as a microscope "grade" polarizer. (Less "extinction" and possibly a non-neutral color shift).

Not sure what the problem is in regards to your darkfield problem. Be sure you have both the field aperture (in the base) and the condenser aperture fully open. You should be OK with the 10X, but it is not uncommon to find it tricky to illuminate (in darkfield) the full area seen by a 4X objective.

dariuskersulis · Post by **dariuskersulis** » Mon Apr 02, 2012 8:49 pm

With all the information you've given me I had to sit down, think about it and play with it. The last time I owned a microscope was when I was 12 maybe. And when I purchased this Optiphot I never really taken a look at he brightfield and went straight to DIC. The view I saw with DIC was pretty good, but didn't like the brightfield, darkfield was a mess as well as phase contrast. So I thought I have to go to the beginning. Aligned the condenser and took a snapshot of the brighfield, and something looked very wrong:

I know the condenser is aligned, but I'm still getting horribly uneven illumination. Then I remembered that I can adjust the bulb position, whalah… I was getting much brighter area in the middle with not as much light fall off on the sides. Switched to darkfield and it looked much improved with illumination across the FOV. But then these horrific light blobs in the middle:

Found a resource for troubleshooting darkfield: http://www.olympusmicro.com/primer/tech ... ouble.html

Near the bottom found a problem "When viewing the specimen through a 10x objective, there is a oblong spot of light in the center of the viewfield." Not exactly oblong spot but it gave a clue that diffuser filter might be needed. I happen to have it all along, just never used it. Slid the filter down and the bokeh spots disappeared. Now I think I have pretty good darkfield image across FOV of 10x objective. Even in phase contrast before I had narrow column of illumination and to the sides the light fell of sharply to complete darkness. Just prior to popping the diffuser I thought I'll never use phase contrast at all as the view plain sucks. Now phase contacts looks simply beautiful. Arguably it may not look as good as DIC in photohgraps but viewing live is just gorgeous. I believe now even DIC has improved alot, I get brighter and more even illuminated image. Just an eye candy now.

Now back to polarized light situation. I understand that I need another, linear polarizer, which I could adjust the angle in relation to the one in the condenser. With pretty much all of my DIY projects I have been disappointed in both quality of workmanship, results and cost, so I think I would be inclined to find dedicated analyzer for Optiphot.

Is this is what I need: http://www.ebay.com/itm/Nikon-Analyzer- ... 519e494fe2

If so I believe it's missing a screw to hold the trinocular head. And then how do I rotate the thing? Anyone familiar with this piece? I've seen those couple of times on ebay already.

Thank you very much!

RogelioMoreno · Post by **RogelioMoreno** » Tue Apr 03, 2012 4:23 am

Darius,

That analyzer is not rotatable, you will need a rotatable polarizer like the following:

http://www.ebay.com/itm/ws/eBayISAPI.dl ... true&rt=nc

Rogelio

dariuskersulis · Post by **dariuskersulis** » Tue Apr 03, 2012 6:06 am

Rogelio,

From the pictures It's not obvious where to mount the rotatable polarizer. My initial thought is to place it over the field lens.

If this is the case then in theory if I were to get the non rotatable analyzer like in this listing:

http://www.ebay.com/itm/Nikon-Analyzer- ... 6437651024

And then simple linear polarizer over the field lens it should work, right? Am I missing something?

Starshade · Post by **Starshade** » Tue Apr 03, 2012 8:37 am

Thank you Charles

Darius: I'm glad that you managed to figure out the alignment issues (hopefully all of them).

In your case DIY should do the job, but the polarizing elements have to be of good quality and desirably come from the same brand to avoid disappointment. At least that's my opinion. The way you attach them (with screws or duct tape) does not matter that much as long as you understand the basics of the technique. Yes, there might be some issues if the polarizers don't have a perfectly horizontal angle, but again, it's possible to sort it out with DIY designs. At least I did that on an occasion, and had perfect results that saved hundreds of dollars.

In the worst case you can lay a polarizer flat somewhere there:

. I just don't know how to install a "properly" rotatable element on your condenser without some modifications.
As long as the polarizer is before the object and you can rotate it one way or another the technique should work.

But one thing for sure - having dedicated gear is awesome.

Btw, you DF image kind of concerned me in terms of chromatic aberrations, they are too strong on the periphery. What aberration correction do your lenses have?

Pau · Post by **Pau** » Tue Apr 03, 2012 11:20 am

dariuskersulis wrote:...I know the condenser is aligned, but I'm still getting horribly uneven illumination.

the posted sample image doesn't seem centered at all. Maybe you need to aling all the illumination components.

To obtain good DF at low magnification you can play with the condensor heigh. With the 4X both for DF and BF may be convenient to remove the top condenser lens

Starshade wrote:...In your case DIY should do the job, but the polarizing elements have to be of good quality and desirably come from the same brand to avoid disappointment.

Good quality is to be recommended, of course (true microscope filters or Edmund's Techspec) but the brand doesn't matter, I usually mix pol filters from Nikon, Zeiss, Olympus and Edmund with perfect results